DI ssI!'ILATIoN OF NONNI TROGENOUS COMPOUNDS BY ACETOBAC'r PASEURIANWI by EL1ELL HACHIRO KA'NASA1I A TîII3IS submitted to OREGON STAT COLLGE in partial fulfillment of the reqireients for the degree of MASTER OF SCIENCE June 1956 fOVJ,D: tssociate rrofessor of Chemistry In charge of ajoa Professor of Chemistry In charge of jor Chairman of Department of Chemistry Chairman of School Groduate Committee Dean of Graduate School Date thesis is presented 3ú /95 Typed by Jean F. nderson ACKNOWLEDQEMNTS I wish to express my sincere gratitude to Dr. Thoo E. King and Pr. Vernon H. Oheldelin for their encouragement, patience, and many helpful ideas during the course of this work. I went to thank paul .\. Litos for his advice and invaluable assistance during this investiration. To Ivarjorie LaSalle, for her suggestions on the method. of lipolo acid assay, I am very grateful. I wish to acknowledge the generous help of Mary Kay Deviin during theae studies, To my other fellow laboratory members, for their patience, advice and asaistaroe, I am greatly indebted. TAflLI OF TTTTS Pegs ITRorT17rxOt I I I I t i a a i a a e a i 3. A!rR1ALsÁNDEtrffODs s... ..ea.s.a. 3 ODntR1t a s ê s s I I I S e e ter1a13 e I a a S I S S S I e 'repoat1onorCo11s a.a.aeae..aa 4 reprt1oìi of Co11.tre : .xtrnotn . 5 .n1yt1ca1Lethoc13 .saa...a.... 5 rct'1n :eternation . a . 6 nomøtrt Teohn1Lues , . a a b aoto1n?orrtion ..e..sa.... .øt1vtion of ;cct&tc , . a e 6 L1ota Acid ¡eaay . a . s b (?T rt s a Q 1' r , . e s * s . :.urvoy of Oxldntlon . a s e s e 9 altric ?oid Ox1thtion . a e a a a 10 yrv10 e1d 0XidtiOfl s a a a a a a C a 11 tiricdonereot ....... , , . 12 cotoin Forrtion . , 13 rei1to Inhibition . a . Lipoiooidtud1ee .......... .. 15 ¿oet&te ot1vct1on a e a a s a a a i e e 16 . 17 . LIST 0F TABLES Pa ge TABLE I . e . 18 TABLEII . e s s e s e s s s s . s s 19 TABLE III . * . 20 TABLEIV . 21 TABLE V . 22 TABLEVI . 23 LIST OF FIGURES FIgure 1 . 24 FIgure 2 . 25 Figure 3 . 26 Figure 4 s s e e i e e s s s s i . s s s s s s 27 DISSThJILATIO! OF NONNITROGTThIOUS COMPOUNDS BY !CETOBACTER PASTEURIANTJ1'7 INTRODUCTION During the past decEde, the use .f microoranisms, prticu1ar1y bacteria, has contributed greatly In eluci- d0ting e number of bioohern1c1 pathways involved in. mammalian metabolism. Utilizing microbes offers many advantages over some other types of b1oio')1cal materi1a. They are vast in number in varied environments, resu1tinj in organisms possessing diversified metabolic pathways. The unicellular organism is a much simpler form of life, possessing a less complicated makeup of biological processes. This is quito useful in that specific reactions in the major pathways such as the tricarboxylic acid cycle and the Embden-ìeyerhof pathway can be studied thoroughly. Sometimes in bacterial systems, a major oxidtiv pathway such as t10 tricarhoxylic acid cycle may show very little sign of activity. The lack of activity of this major pathway may lead to discovery of other important biological pathways. This has been shown in cetobacter suboxydans, (an organism without tricarboxylic acid cycle activity) by demonstrating the pentose cycle in toto (5, 11-26). 2 An approacIi in studying these unusual organisis is by investigating a related species possessing the miss- ing naor pathway. Studies in this direction has been started in this work with the species, Pcetobacter st- eurianum, an organisai showing tricarboxylic acid cyclo activity. 3 MÂTEflI ALS AITD METHODS Organi sus Acotobactor pasteurianum ATCC 6033 and Acetobacter suboxydans ATOO 621 wore obtained from tho American Typo Culturo Collection. Transfers rere made veokly on rlycer- ol-yeast oxtract acar slants. Streptococcus Caecal±s 10Cl vas kindly furnished by Dr. I. C. nsa1us. This organism was kept on stab agar tubes. Transfers were made froi the stock culture tube when needed. Ilate ri als CoA1(75 purIty), TPN1, DPN1, ATP' and glucose-6- phosphate 'vero obtained from Pabst LaboratorIes . ?yruvic acid (sodium salt), glutathlone, and fruetosc-1,6-dios- pliate iore purchased frotia Schwartz Laborstorios . Dihy- droxyacetone, oxalrìcetio acid and g1ucose-l-phoshate wore -j The following abbreviations aro used: ATI> adonosine triphos hato BAL 2,3-dercapto-1-propano1 OPE cell-free extract C0A coenzyme A DPN dipho8phopyridno nue le et ido ,iLi microic10 PCA tricarboxylic acid triphosphoyridino nuclootide tris tris (hydroxymethyl) arainoniethano M products from Krishell Laboratries. The other materials were purchased as follows: cocarboxylase: S. A. F. Hoffm.ann-Lafloche & Company, PAL1: ann esecrch Laboratories, Trypsin (powder form, 1:110): Pfanstieh]. Chemical Company, cytochrome-C: Sigma Chemical Company, -1etog1utaric acid: General Biochemical moor- porated, and Fleietimann's yeast extract type 3: Standard Brands Incorporated. Pret,aration of Cells Cultivations of -. pasteurianum and of . sub- oxydans were raudo in 20 liter carboys containing 10 liters of medium with the following composition: glycerol, 5, yeast extract, l, K2ID?04, 0.5%, and snl1 uantitios of Dow anti-foam; pH 60. The inocula for the medii.tm were prepared by trans- ferring cells from agar slants to 500 ml. Erlenmeyer flasks containing 100 mi. of the above medium. They were placed on shakers at room temperature for 36 hours of growth. Ten liters of medium were inocuL.ted with 200 ¡nl. of actively growing cells. The inoculated batch was placed in a 30 C. incubator and wea aerated vir;orously with star- ilized air. Aftor 48 hours of growth, the culture ':as checked for contamination by microscopic and plate methods. Cells were separated on a Sharples centrifuge. They were 5 suspended into 300 ml. of 0.05 phosphate buffer, pH 6.0, and shaken for one hour at room temperature. The cells were then collected by contrifu icn on a ervE,ll centri- fuge and washed twice by resuspension in distilled water. The final usronsjon of cells was dried by lyophllization. Dry cells were stored at _1000. Yields ranged from 5 to 8 gramas per ten liters of m6dium. Preparation of Cell-free ttracts 0F1' were prepared as follows: 2 gin. of lyophil- ized cells were suspended in 40 ml. of glass distilled vter or 0.01 M phosphate buffer, pH 7.4 and treated at sonic frequencies in a Raytheon 200-watt 10-ko. mngneto- striction osci11tor. The cylindrical stainless steel cup containing the cell suspension was cooled by circula- ting water at 6-10° . into the cooling jacket. The cell suspension was treated for a total of fifteen minutes (three five minute periods with two five minute inter- missions) in order to keep the stainless steel cup from overheating. The sonic treated cell suspension was then centri- fuged for 90 minutes at 20,000 x . The solid phase was discarded and the supernatant liuid was stored at _100 C. nalytical ethods rotein Teternainatlon. rote1n was determined by the modified eichse1b8um's iiethod usine crystalline eg albumin as the standard (14, pp. 16-20). Manometric Tecinigues. Oxygen consumption or carbon dioxide formation was determined by conventional techniques using a circular :rhurg apparatus. oetoin Formation. cetoin was determined by the method of Westorfeld (15, pp. 495-502). Blanks were run for each determination. ctivation of %cetate. The method of Kitos et al. (9, pp. 236-237) was used ',':ith slight modifications. In a small test tube, a mixture of glutathione, 20 ,jf, and CoA, 2,iM, was incubated for ten minutes to keep the Coi in the reduced condition. AT, 2OjiM, ?4gCl2, 20 hydroxylamine, 200 )JY, and tris1, 100 yM, potassium ace- tate, 200 )IM, OFE, 11 mg. protein, and water were then added to 2 nil. Subsequent steps were the sanie as those of Kitos et al. (9, pp. 236-237). lipoic Acid Assay. Lipoic acid was determined according to Gunsalus, Polin, and Struglia (4, pp. 849- 857) with modification. Fleischnaann's yeast extract type 3 was used as the standard. The response from 1 mg. of yeast extract was used as one unit of lipoic acid. Earlier work in this laboratory (10) found that in employing the assay system described by C-unsalus et al. (4, pp. 849-857), it was difficult to obtain a standard curve comparable to the one obtained by the publiahed nethod. Thus, results were erratic. By modifyinc, the preparation of the enzyrnatically Uigested casein, standard curves with nore spread were obtainable. By doubling the concentration of the enzytne-dlgested casein, consistent results were always obtained. It was found that the preparation of enzymatic digested casein and Its concentration was critical. The following niethod of digestion proved satisfactory: 50 gni. of vitamin-free casein were added to 490 ml, of water and the mixture was placed on a shaker for 15 minutes. The solution was adjusted to pH 8.0; 10 ini. of 51 trypsln solution was added, fter the mixture vies shaken for 10 minutes, It was transferred to a 1000 ial, flask and placed 370 In a C. bath. J thin layer of toluene was added. The flask was fitted with a stirrer and the mixture was very slowly stirred. fter 24 hours of incuhtIon, the contents were transferred to a large separatory funnel and the toluene removed. The mixture was adjusted to pT 6.0 and filtered with mild suction trourh a layer of cotton on a Buchnor funnel. 25 gm. of Norite were aded to the mixture and shaken for 30 minutes. The Norite was removed by fil- tration and the digested mixture wa adjusted to pH 3.8.